CN103320339B - Leuconostoc mesenteroides strain - Google Patents
Leuconostoc mesenteroides strain Download PDFInfo
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- CN103320339B CN103320339B CN201310077194.0A CN201310077194A CN103320339B CN 103320339 B CN103320339 B CN 103320339B CN 201310077194 A CN201310077194 A CN 201310077194A CN 103320339 B CN103320339 B CN 103320339B
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Abstract
The invention relates to the field of biological technology, and discloses an application of a leuconostoc mesenteroides strain, wherein the strain is preserved in China Center for Type Culture Collection (CCTCC), the preservation date is Nov. 26, 2012, and the preservation number is CCTCC M 2012479. The strain has good inhibition effect on peronophythora litchi and/or anthracnose pathogen, can substantially reduce activities of polyphenol oxidase (PPO) and/or peroxidase (POD) of lichee or longan, and keep superoxide dismutase (SOD) activity of pericarps. Therefore, the strain has important application value in fresh-keeping aspect of fruits such as lichee, longan and the like.
Description
Technical field
The present invention relates to biological technical field, more specifically, relate to a strain Leuconostoc mesenteroides bacterial strain.
Background technology
Before fresh fruit of vegetables is eaten from gathering by human consumer, because self respiration and causal organism infect, a large amount of fruits and vegetables are had to rot to lose transport, storage after gathering every year.Country's agricultural products fresh-keeping Engineering Technical Research Centre investigation finds, the fruits and vegetables that China produces per year at present are every year from field to dining table rate of loss up to 25 ~ 30%, and year loses nearly 80,000,000,000 Renminbi.Therefore postharvest technology of fruits and vegetables development research is carried out further, very urgent to ensure making full use of of the realization of fruit and vegetable food added value and resource.
Meanwhile, along with growth in the living standard, the requirement of people to food such as fruit is more and more higher, requires on basis at the color for fruit, the safety and environment protection of what people started to pay close attention to more and more is food.
Fruit preservation method international at present roughly has two kinds.One is ethylene absorbent, normally absorbs ethene with venting bags, makes fruit can not be ripe further, harmless without medicine, but fresh keeping time and effect instability.Another kind is waxing, and a lot of fruit self can produce fruit wax, preventable disease worm, microorganism encroach, but store, transport fruit in season wax easily rubbed part, effect can not ensure, and inevitably causes human consumer to eat fruit wax.Appropriate sulfurous gas has good fresh-keeping effect to the fruit such as lichee, longan, but excessive use causes residual exceeding standard, and not only nutrient quality is influenced, and eater also there will be the symptoms such as dizziness, Nausea and vomiting and diarrhoea, even can damage hepatic and renal function.Have and report that the most effective means controlling postharvest disease of fruits and vegetables is that refrigeration is in conjunction with chemical bactericide process.But because chemical bactericide remains harm humans health, and phytopathogen easily develops immunity to drugs to chemical bactericide, application is more and more restricted.
The good fruit lichee in the south of the Five Ridges liked for people, litchi fruits is nutritious, its color tool is good, there is the laudatory title of " fruit king ", has Development volue, have good economic benefit, be one of the fruit of China's most competitive power in the international market, the maximum country of lichee is cultivated at present in the world by China.But lichee is difficult to preserve, fresh-keeping for lichee, have the summary of " sunlight becomes, and two fragrant becomes, and taste became on 3rd, and color, smell and taste were gone to the greatest extent on 4th ".The picking fruit of lichee to be in of short duration and summer of sweltering heat season, after adopting, self respiration is vigorous, water evaporation rate is high, under the dual function that self enzyme and extraneous pathogenic bacteria are attacked, very easily brown stain is rotted, not easily preserve, not storage tolerance, seriously limits finding a good sale in and outlet of lichee, constrains the developing of regional market.According to statistics, the loss that lichee causes because rotting every year accounts for more than 20% of ultimate production.
At present, the storage technique of domestic and international litchi fruits mainly contains physics storage and fresh chemically two kinds.Conventional antiseptic drug mainly benomyl, TBZ, P applied levels, sterilizing prestige, derosal etc. in the fresh chemically process of litchi fruits, if application number is the patents such as CN200610123793.1, CN03140439.1, CN201010127794.X; Comprise the chemical treatments such as sulphuring or sulfiting in addition, such as application number is the patents such as CN96122329.4, CN97105377.4 and CN00130815.7.Although chemical agent can more effectively play anticorrosion, fresh-keeping effect, its residual contamination produced not only affects local flavor, and the most important thing is residual have potential threat to HUMAN HEALTH.
It is neural that chemical preservative touches the human consumer enjoying food-safety problem to decoct repeatedly.In addition spray the report such as sour lichee, lime mango to be reported in media and network, constantly cause the uneasiness of human consumer.Find the biological way of keeping fresh of safety non-toxic, for substituted chemistry fresh-keeping method, become the focus of concern.Microbe species is abundant, pathways metabolism is various, and can produce active substance nature disease bacterium being had to antagonistic action.Therefore, utilize microorganism and preparation thereof to carry out fruit freshness preserving, can reach nontoxic, harmless, noresidue, free of contamination requirement, eliminate the residual of chemical agent safely and effectively, meet the development trend of food safety.
Summary of the invention
Technical problem to be solved by this invention is the deficiency overcoming existing biological fruit and vegetable preservation technique, provides a strain Leuconostoc mesenteroides bacterial strain, for the biological way of keeping fresh field of fruits and vegetables provides effective new strains.
Another object of the present invention is the cultural method of described bacterial strain.
Object of the present invention is realized by following technical proposals:
Provide a strain Leuconostoc mesenteroides (Leuconostoc Mesenteroides) bacterial strain, described bacterial strain is in China typical culture collection center (CCTCC) preservation, and preservation date is on November 26th, 2012, and preserving number is CCTCCM2012479.The address of described China typical culture collection center is Luojiashan, Wuchang, Wuhan City, Hubei Province.Described Strain Designation is: Leuconostoc mesenteroides dgnkzx002LeuconostocMesenteroides dgnkzx002.
What the present inventor produced from orchard, research of agricultural science center, Dongguan collects described bacterial strain without disease and pest, the longan fruit of healthy high-quality that has no mechanical damage.
Particularly, fruit rind is torn into suitable fritter, with hand pulp is squeezed and rottenly make fruit juice all extrude (whole process is all aseptic technique) to be put in previously prepd sterile flask, to be placed in 37 DEG C of anaerobic culture boxes and to carry out spontaneous fermentation cultivation.Fermentation culture, after 2 days, pipettes fermented liquid and adopts series concentration gradient dilution method to dilute, be diluted to 10
-1, 10
-2, 10
-3, 10
-4, 10
-5, 10
-6, in triplicate.Get 10
-4, 10
-6the diluent of two gradients, draws 0.2 ~ 0.3mL and is added on the MRS substratum that solidified, then rapidly that fermented liquid is evenly spreadable with spreading rod.Then flat-plate inverted is placed in 37 DEG C of anaerobic culture boxes to cultivate.Treat that bacterium colony grows, the obvious single bacterium colony of picking colony morphological differences enters plate streaking and is separated, and line separation is carried out purifying 3 times and carries out gram stain microscopy.
The moiety of described MRS substratum is: 1L distilled water, peptone 10g, extractum carnis 10g, yeast extract paste 5g, dipotassium hydrogen phosphate 2g, dibasic ammonium citrate 2g, sodium acetate 5g, glucose 20g, magnesium sulfate heptahydrate 0.58g, four water manganous sulfate 0.25g, tween-80 1mL, calcium carbonate 15g, agar 18g, pH6.2 ~ 6.4,121 DEG C of sterilizing 15min.
Invention also provides the cultural method of described bacterial strain: be by the glycerine pipe freezing bacterial classification of Leuconostoc mesenteroides (LeuconostocMesenteroides) dgnkzx002 by 2% inoculum size in 20mLMRS liquid nutrient medium, activation culture obtains seed liquor; By seed liquor according to 5% inoculum size receive in 1000mLMRS liquid nutrient medium, cultivation and fermentation, fermentation liquor 8000r/min, 4 DEG C of collected by centrifugation thalline, after stroke-physiological saline solution washing, centrifugal and get final product.
Described MRS liquid culture based component: 1L distilled water, peptone 10g, extractum carnis 10g, yeast extract paste 5g, dipotassium hydrogen phosphate 2g, dibasic ammonium citrate 2g, sodium acetate 5g, glucose 20g, magnesium sulfate heptahydrate 0.58g, four water manganous sulfate 0.25g, tween 80 1mL, pH6.2 ~ 6.4,121 DEG C of sterilizing 15min.
The biological morphology feature of described Leuconostoc mesenteroides bacterial strain: colonial morphology is on MRS substratum purifying flat board: circular, creamy-white, convex growth, moistening; Microscopic examination thalli morphology is: the positive coccobacillus of Gram.
Described Leuconostoc mesenteroides has good restraining effect to peronophythora litchi, anthrax bacteria, and this bacterial strain obviously can also reduce polyphenoloxidase and/or the Peroxidase activity of lichee or longan, keep pericarp superoxide-dismutase (SOD) active.Therefore, this bacterial strain has important using value at the fresh-keeping aspect of the fruit such as lichee or longan.
The invention provides one preferably utilisation technology scheme, Leuconostoc mesenteroides is prepared into a kind of preserving fungus agent, is effective activeconstituents with the thalline suspension of Leuconostoc mesenteroides (Leuconostoc Mesenteroides) bacterial strain; Described preserving fungus agent comprises each component of following weight percentage:
Chitosan 0.5% ~ 1.5%;
Citric acid 2% ~ 3%;
Sodium.alpha.-hydroxypropionate 1% ~ 5%;
Kojic acid 0.5% ~ 1%;
Sodium-chlor 0.85% ~ 0.9%;
Water surplus;
In 100% system of above-mentioned each component composition, add the thalline suspension of described Leuconostoc mesenteroides (LeuconostocMesenteroides) bacterial strain, make its ultimate density in system be 10
8~ 10
9cFU/mL.
More preferably, described preserving fungus agent comprises each component of following weight percentage:
Chitosan 1%;
Citric acid 2%;
Sodium.alpha.-hydroxypropionate 1%;
Kojic acid 0.5%;
Sodium-chlor 0.85%;
Water surplus;
The application of the preserving fungus agent of Leuconostoc mesenteroides, above-mentioned preserving fungus agent is applied to fruit can obtain fresh-keeping effect well, is especially applied to the good fruit lichee in the south of the Five Ridges or longan, can obtains best fresh-keeping effect.
The present invention is by experimental summaries a large amount of for a long time, and described preserving fungus agent all can obtain fresh-keeping effect well to lichee and longan.In the fresh-keeping test of the kind longans such as the kind lichee such as Litchi chinensis cv. Nuomici, the glutinous lichee of Jing Ganghong and No. two, Dongfeng, effect is the most outstanding.
Preferably, the application of the preserving fungus agent of described Leuconostoc mesenteroides is specially to be applied to and suppresses anthrax bacteria and/or peronophythora litchi.
Preferably, the application of the preserving fungus agent of described Leuconostoc mesenteroides is specially the polyphenoloxidase and/or peroxidase that are applied to and suppress lichee.
Preferably, the application of the preserving fungus agent of described Leuconostoc mesenteroides is specially to be applied to and keeps pericarp superoxide-dismutase (SOD) active.
Described bacterial strain is applied to preserving fruit and vegetable utilizing and has good effect, is especially applied to the fresh-keeping of lichee or longan.To fruit postharvest diseases such as common anthrax and peronophythora litchis, there is remarkable, stable fungistatic effect.To lichee polyphenol oxydase and peroxidase, also there is restraining effect well simultaneously; To pericarp superoxide-dismutase (SOD) activity, there is significant maintenance effect.
The present invention has following beneficial effect:
The invention provides the new Leuconostoc mesenteroides of a strain (Leuconostoc Mesenteroides) dgnkzx002 bacterial strain, biological way of keeping fresh field for fruits and vegetables provides a new microorganism member, prove through test, described bacterial strain has significant effect to the fresh-keeping of fruits and vegetables.Especially to peronophythora litchi, lichee anthrax bacteria, there is good restraining effect, and this bacterium obviously can also reduce polyphenoloxidase and the Peroxidase activity of fruit, keep pericarp superoxide-dismutase (SOD) active.
The present invention is based on new Leuconostoc mesenteroides (Leuconostoc Mesenteroides) dgnkzx002 bacterial strain, the biological way of keeping fresh field for fruits and vegetables provides a kind of new preserving fungus agent.Described preserving fungus agent especially has good restraining effect to peronophythora litchi, anthrax bacteria, and this bacterium obviously can also reduce polyphenoloxidase and the Peroxidase activity of fruit, keeps pericarp superoxide-dismutase (SOD) active.
The present invention is on the basis of Leuconostoc mesenteroides (Leuconostoc Mesenteroides) the dgnkzx002 bacterial strain obtained, by compatible for the thalline suspension of chitosan, citric acid, Sodium.alpha.-hydroxypropionate, kojic acid, sodium-chlor and described bacterial strain, obtain excellent preserving fruit and vegetable utilizing effect.When selecting microbial inoculum auxiliary material, the present inventor combines the character research to various auxiliary material through great many of experiments, summary obtains technical solution of the present invention: chitosan is a kind of polysaccharose substance of safety non-toxic, it can not be utilized by Institute of Micro-biology, therefore, it is possible to suppress the growth of some pathogenic bacterias, by the important component one of of chitosan as the carrier of thalline suspension of the present invention, one deck can be formed on pericarp surface and there is water conservation, the preservative film of controlled atmosphere effect, and used fresh-keeping of the present invention is facultative anaerobe with bacterium, keep active for a long time in the low-oxygen environment that can be formed at chitosan, thus the fresh-keeping system that formation one is good, citric acid can provide suitable sour environment, contributes to the dissolving of chitosan, is also conducive to the content keeping anthocyanin, is finally conducive to the maintenance of fruit colour, kojic acid is a kind of organic acid produced through some fungi aerobic fermentation, extensively exists with fermented product, has anti-oxidant and bacteriostatic action, can suppress the brown stain of lichee, in microbial inoculum system provided by the invention, the fresh-keeping of Sodium.alpha.-hydroxypropionate and water retention property can be played, through constantly analysis and research and great many of experiments are summed up, under the system that suitable compatibility provided by the invention is formed, bacterial strain can survive well and play active function, and importantly thalline suspension system of the present invention creates significant utilisation technology effect jointly.
The present invention is on the basis of Leuconostoc mesenteroides (Leuconostoc Mesenteroides) the dgnkzx002 bacterial strain obtained, further summary obtains the reasonable compatibility ratio of each component, in suitable amount ranges, preserving fungus agent of the present invention has good fresh-keeping effect, is applied to
licheeor extending longan shelf life, storage
2vivid outward appearance and good local flavor still can be kept after 5 days
, healthy fruit can reach 86.7%; In addition, preserving fungus agent safety non-toxic of the present invention, have good water retention property, soluble in water be convenient to edible before the advantage such as cleaning.
The present invention applies described bacterial strain and prepares microbial inoculum and successfully realize application, and the activity application overcoming usual microbial bacteria is confined to the defect in laboratory, has good actual application prospect.Described bacterial preparation process is simple, and application is convenient, provides strong technology enlightenment and instruct for the application of microbial bacteria in preserving fruit and vegetable utilizing, has important using value and actually applies feasibility.
Accompanying drawing explanation
Fig. 1. Leuconostoc mesenteroides dgnkzx002 bacterial strain colonial morphology (MRS substratum, 37 DEG C, 2 days).
Fig. 2. Leuconostoc mesenteroides dgnkzx002 gram stain microscopy result.
Fig. 3 anthrax bacteria suppresses contrast.
Fig. 4. Leuconostoc mesenteroides dgnkzx002 is to anthrax bacteria inhibition figure.
Fig. 5. peronophythora litchi suppresses contrast.
Fig. 6. Leuconostoc mesenteroides dgnkzx002 is to peronophythora litchi inhibition figure.
Fig. 7. Leuconostoc mesenteroides dgnkzx002 is to POD activity influence design sketch.
Fig. 8. Leuconostoc mesenteroides dgnkzx002 is to PPO activity influence design sketch.
Fig. 9. the comparison diagram that the red glutinous denaturation progression in well hilllock of reference examples and embodiment 2 changes with storage time.
Figure 10. the correlation curve figure that the Litchi chinensis cv. Nuomici healthy fruit of reference examples and embodiment 3 changes with storage time.
Figure 11. the longan healthy fruit after Leuconostoc mesenteroides dgnkzx002 process.
Figure 12. Leuconostoc mesenteroides dgnkzx002 is to longan peel PPO activity influence effect.
Figure 13. Leuconostoc mesenteroides dgnkzx002 is to longan peel POD activity influence effect.
Figure 14. Leuconostoc mesenteroides dgnkzx002 is to longan peel SOD activity influence effect.
Figure 15. the healthy fruit (commodity rate, %) of longan fruit after preservation agent process of the present invention.
Figure 16. the rate of weight loss (%) of longan fruit after preservation agent process of the present invention.
Figure 17. soluble solid (TSS) content of longan fruit after preservation agent process of the present invention.
Figure 18. the Vitamin C content of longan fruit after preservation agent process of the present invention.
Figure 19. the reducing sugar content of longan fruit after preservation agent process of the present invention.
Figure 20. the total sugar content of longan fruit after preservation agent process of the present invention.
Figure 21. to longan peel PPO activity influence effect after preservation agent process of the present invention.
Figure 22. to longan peel POD activity influence effect after preservation agent process of the present invention.
Figure 23. to longan peel SOD activity influence effect after preservation agent process of the present invention.
Embodiment
The present invention is elaborated further below in conjunction with the drawings and specific embodiments.Following examples of the present invention are the present invention's preferably embodiment, the present invention mainly sets forth described bacterial strain and the application thought based on described bacterial strain, in embodiment, the replacement of simple parameter can not repeat one by one in an embodiment, but therefore do not limit the present invention, change, the modification done under other any does not deviate from spirit of the present invention and principle, substitute, combine, simplify, the substitute mode of equivalence should be regarded as, be included in the present invention.
Embodiment 1
The present inventor on September 11st, 2011 gather from the longan fruit that is produced from orchard, research of agricultural science center, Dongguan healthy, without disease and pest, the longan fruit that has no mechanical damage, the peeling of 150g fruit, fragmentation are put in spontaneous fermentation in previously prepd sterile flask, in 37 DEG C of anaerobic culture boxes, carry out fermentation culture 2 days with pericarp by after pulp crushing together with fruit stone.Ferment after 2 days, pipette fermented liquid and adopt series concentration gradient dilution method to dilute, with sterilized water, fermented liquid is diluted to 10
-1, 10
-2, 10
-3, 10
-4, 10
-5, 10
-6the diluent of totally 6 gradients, in triplicate.Get 10
-4, 10
-6the diluent of two gradients, draws 0.2 ~ 0.3mL and is added on the MRS substratum that solidified, then rapidly that fermented liquid is evenly spreadable with spreading rod.Then flat-plate inverted is placed in 37 DEG C of anaerobic culture boxes to cultivate.Treat that bacterium colony grows, the obvious single bacterium colony of picking colony morphological differences enters plate streaking and is separated in MRS substratum purifying flat board, and line separation carries out purifying 3 times.
On MRS substratum purifying flat board, colonial morphology is: circular, creamy-white, and convex growth is moistening, sees shown in accompanying drawing 1.
The moiety of described MRS substratum is: the moiety of described MRS substratum is: 1L distilled water, peptone 10g, extractum carnis 10g, yeast extract paste 5g, dipotassium hydrogen phosphate 2g, dibasic ammonium citrate 2g, sodium acetate 5g, glucose 20g, magnesium sulfate heptahydrate 0.58g, four water manganous sulfate 0.25g, tween-80 1mL, calcium carbonate 15g, agar 18g, pH6.2 ~ 6.4,121 DEG C of sterilizing 15min.
By described bacterial strain in China typical culture collection center (CCTCC) preservation, preservation date is on November 26th, 2012, and preserving number is CCTCCM2012479.The address of described China typical culture collection center is Luojiashan, Wuchang, Wuhan City, Hubei Province.Described Strain Designation is: Leuconostoc mesenteroides dgnkzx002Leuconostoc Mesenteroides dgnkzx002.
Gramstaining is tested
S1. smear: get a slice totally without oily slide glass, sterilized water is dripped in centre wherein, then to get with aseptic technique method with transfering loop and has cultivated bacterial strain a little, be placed in the water droplet of slide glass and smoothen, and notices that bacterium amount should not be too much, otherwise, not easily see single thalline clearly.
S2. fix: by the slide that coats on spirit lamp flame quickly through 3 ~ 4 times, make water evaporates, now microorganism is close on slide.
S3. just contaminate: add ammonium oxalate crystal violet staining agent 1, dye 1 minute.
S4. mordant dyeing: incline dye liquor rinsing gently with washing bottled water, adds iodine staining 1 minute, washing, then blots with thieving paper.
S5. decolour: slide glass is slightly tilted, drip 95% alcohol decolouring 20 ~ 30 seconds (be washed till in the alcohol flowed down without during purple till), then wash.
S6. redye: on slide, drip sarranine dye liquor about 2 minutes, washing, then blots with thieving paper.Should prevent dye liquor from drying up in dyeing.
S7. microscopy: carry out microscopy with oily mirror after drying, microscopy is shown as a strain Gram-positive coccobacillus.Thalli morphology the results are shown in accompanying drawing 2.
Bacteriostatic test:
Because anthrax and peronophythora litchi adopt rear lichee and the modal disease of longan, therefore the present embodiment selects anthrax bacteria, peronophythora litchi as cause of disease indicator.
S1. spore suspension is prepared:
The bacterial classification of the glycerine pipe freezing of the Leuconostoc mesenteroides dgnkzx002LeuconostocMesenteroides dgnkzx002 of 50% glycerine pipe freezing is pressed 2% inoculum size in 20mLMRS liquid nutrient medium, and leave standstill activation culture 20 hours in 37 DEG C of incubators; The seed liquor activated is received in 1000mLMRS liquid nutrient medium by 5% inoculum size, and in 37 DEG C of incubators quiescent culture 20 hours; After having fermented, fermentation liquor 8000r/min, 4 DEG C of centrifugal 15min collect thalline, wash 2 times, the viable bacteria body needed for centrifugal acquisition by stroke-physiological saline solution.Finally with appropriate physiological saline thalline hanged and made cell suspending liquid.
MRS liquid culture based component: 1L distilled water, peptone 10g, extractum carnis 10g, yeast extract paste 5g, dipotassium hydrogen phosphate 2g, dibasic ammonium citrate 2g, sodium acetate 5g, glucose 20g, magnesium sulfate heptahydrate 0.58g, four water manganous sulfate 0.25g, tween 80 1mL, pH6.2 ~ 6.4,121 DEG C of sterilizing 15min.
S2. for anthrax bacteria adopt improvement agar diffusion method: the first agar content of falling one deck 1.5%(mass percentage content on aseptic flat board) PDA substratum (PDA substratum is conventional formulation, concrete composition is: 200g potato decortication is cut into small pieces, after adding the soft filtered through gauze of poach, 20g sucrose and 15g agar is added in filtrate, finally add water and mend to 1000mL, nature pH, 121 DEG C of sterilizing 15min), draw the spore suspension 1ml prepared, join and melt and be cooled to the middle mixing of the 6mlPDA substratum of about 50 DEG C (agar mass percentage content is 0.9%), topple on the PDA flat board solidified.After the substratum cooled and solidified of upper strata, it is 6mm hole that flat board is beaten some diameters, respectively by cell free supernatant fermented liquid, containing cell fermentation liquid and be added in different holes with somatic cells suspension prepared by physiological saline and carry out bacteriostatic test, often 3 repetitions are established in process, take sterilized water as contrast, flat board is placed in 28 DEG C of constant incubators to cultivate after 2 ~ 3 days and observes antibacterial situation and inhibition zone size.Experimental result is shown in shown in accompanying drawing 3 and accompanying drawing 4, and wherein accompanying drawing 3 is contrast, and accompanying drawing 4 is the bacteriostatic experiment result of dgnkzx002.
S3. for the dull and stereotyped face-off method of peronophythora litchi PDA: first move in PDA culture medium flat plate central authorities the agar block that a diameter is about the mould indicator of white epidemic disease of 2cm, owing to first allowing its growth 2 ~ 3 days after the longer culture transferring of the mould growth cycle of white epidemic disease, then the place punching of 5mm is about at the edge that falls apart from white phytophthora, cell free supernatant fermented liquid is injected respectively in different holes, bacteriostatic test is carried out containing cell fermentation liquid and with somatic cells suspension prepared by physiological saline, each process repeats with sterilized water for 3 times to be contrast, assay plate is placed in 28 DEG C of incubators to cultivate 3 ~ 5 days, day by day observe, measure inhibition zone radius, experimental result is shown in shown in accompanying drawing 5 and accompanying drawing 6, wherein accompanying drawing 5 is contrast, accompanying drawing 6 is the bacteriostatic experiment result of dgnkzx002.
S4. inhibitory enzyme is lived and is tested
Because polyphenoloxidase (PPO) and peroxidase (POD) are most important two kinds of enzymes in lichee and longan browning, therefore, experiment selects PPO and POD to carry out enzyme to live inhibition test.
The preparation of crude enzyme liquid:
Get Fructus Litchi 2g, add the grinding of 0.4gPVP ice bath, 4 DEG C, the centrifugal 15min of 9500r/min with the phosphoric acid buffer of the 0.2MpH6.8 of 5 times amount, collect supernatant liquor and be crude enzyme liquid, 4 DEG C save backup.
The phosphoric acid buffer of POD: reaction system comprises: 2175 μ LpH6.8,375 μ L0.08%(volume by volume concentrations) H
2o
2solution, (the dgnkzx002 bacterial classification of 50% glycerine pipe freezing ferments 2 days in MRS liquid nutrient medium by 5% inoculum size 325 μ L inhibitor, and after having fermented, fermentation liquor 9500r/min, 4 DEG C of centrifugal 15min collect thalline, wash 2 times by 0.9% stroke-physiological saline solution, 4 DEG C of placements are for subsequent use.Be 10 with cell concn prepared by 0.9% physiological saline respectively
6~ 10
9cFU/mLdgnkzx002 thalline suspension, cell free fermentation supernatant liquor, be 10 containing cell concn
6~ 10
9cFU/mL fermented liquid is tested as inhibitor, the phosphoric acid buffer of control group 0.2MpH6.8 replaces), 750 μ L0.05M methyl catechol solution, 75 μ L dilute the above-mentioned crude enzyme liquid of 10 times, light absorption value is measured at 470nm place, timing from above-mentioned enzyme liquid adds, an every 10 seconds records light absorption value, calculates enzyme with initial straight slope and lives.An enzyme unit definition alive is: under condition determination, light absorption value per minute increases the enzyme amount needed for 0.001, enzyme replicate(determination) alive three times.Dgnkzx002 is shown in shown in accompanying drawing 7 POD activity influence effect.
PPO activity determination method: reaction system comprises: the phosphoric acid buffer of 2550 μ L0.2MpH6.8 (consists of: Sodium phosphate dibasic and SODIUM PHOSPHATE, MONOBASIC, with reference to ordinary method preparation), 750 μ L0.2M pyrocatechol solution (consisting of: pyrocatechol solution prepared by above-mentioned phosphate buffered saline buffer), (the dgnkzx002 bacterial classification of 50% glycerine pipe freezing ferments 2 days in MRS liquid nutrient medium by 5% inoculum size 325 μ L inhibitor, after having fermented, fermentation liquor 9500r/min, 4 DEG C of centrifugal 15min collect thalline, 2 times are washed by 0.9% stroke-physiological saline solution, 4 DEG C of placements are for subsequent use.Be 10 with cell concn prepared by 0.9% physiological saline respectively
6~ 10
9cFU/mLdgnkzx002 thalline suspension, cell free fermentation supernatant liquor, be 10 containing cell concn
6~ 10
9cFU/mL fermented liquid is tested as inhibitor, and the control group phosphoric acid buffer of aforementioned 0.2MpH6.8 replaces), the above-mentioned crude enzyme liquid of 75 μ L.Measure light absorption value at 398nm place, timing from above-mentioned enzyme liquid adds, an every 10 seconds records light absorption value, calculates enzyme with initial straight slope and lives.An enzyme unit definition alive is: under condition determination, light absorption value per minute increases the enzyme amount needed for 0.001, enzyme replicate(determination) alive 3 times.Dgnkzx002 is shown in shown in accompanying drawing 8 PPO activity influence effect.
Leuconostoc mesenteroides dgnkzx002 to peronophythora litchi and anthrax bacteria inhibited, also have well inhibiting to lichee polyphenol oxydase and lichee peroxidase simultaneously.
Embodiment 2
S1. the cultivation of fresh-keeping bacterium:
S11. the activation of bacterial strain: fresh-keeping bacterial strain is pressed 2% inoculum size in 20mLMRS liquid nutrient medium by glycerine pipe, and leave standstill activation culture 20 hours in 37 DEG C of incubators;
MRS liquid culture based component: 1L distilled water, peptone 10g, extractum carnis 10g, yeast extract paste 5g, dipotassium hydrogen phosphate 2g, dibasic ammonium citrate 2g, sodium acetate 5g, glucose 20g, magnesium sulfate heptahydrate 0.58g, four water manganous sulfate 0.25g, tween 80 1mL, pH6.2 ~ 6.4,121 DEG C of sterilizing 15min.
S12. enlarged culturing: by the seed liquor activated by 5% inoculum size be inoculated in 1000mLMRS liquid nutrient medium, and in 37 DEG C of incubators quiescent culture 20 hours;
S13. thalline suspension preparation: after having fermented, fermentation liquor 8500r/min, 4 DEG C of centrifugal 15min collect thalline, wash 2 times, the viable bacteria body needed for centrifugal acquisition by stroke-physiological saline solution.Finally with appropriate physiological saline thalline hanged and made cell suspending liquid, cell concentration 2.4 × 10
8cFU/mL.
S2. test for trying material with the red glutinous lichee in well hilllock.Pluck litchi fruits morning at fine day, choose healthy homogeneous without disease and pest, size, have no mechanical damage, the litchi fruits of about 8 maturations; Transport laboratory back rapidly, carry out germicidal treatment with ultra violet lamp 30min; With above-mentioned bacterium liquid even application on litchi fruits surface, with bubble chamber splendid attire after normal temperature dries, and at the inner place mat newspaper of case, in order to avoid Fructus Litchi is caused brown stain by the globule dipping that respiration produces on tank wall, 30 ± 2 DEG C, preserve under the hot and humid condition of relative humidity 85% ~ 90%, duration of storage can observe experimental group color significantly better than control group.Result as shown in Figure 9.
Reference examples:
Choose healthy homogeneous without disease and pest, size, have no mechanical damage, the litchi fruits of about 8 maturations; Germicidal treatment is carried out with ultra violet lamp 30min; With tap water even application on litchi fruits surface, with the bubble chamber splendid attire of liner newspaper after normal temperature dries, 30 ± 2 DEG C, preserve under the hot and humid condition of relative humidity 85% ~ 90%.
Embodiment 3: preservation agent application experiment
Under determining that bacterial strain has the prerequisite of freshening effect, the present invention is effective activeconstituents with the thalline suspension of Leuconostoc mesenteroides bacterial strain, determine simultaneously the biological substance rationally matched composite go out a kind of safety, efficiently bio-preservative.Described preserving fungus agent comprises each component of following mass percentage: 1% chitosan, 2% citric acid, 0.85% sodium-chlor, 0.5% kojic acid and 2% Sodium.alpha.-hydroxypropionate, and surplus is water; In 100% system of above-mentioned each component composition, add the thalline suspension of described bacterial strain, make its cell concentration in system be 2.9 × 10
8cFU/mL, preparation obtains a kind of preservation agent.
S1. the cultivation of fresh-keeping bacterium:
S11. the activation of bacterial strain: the glycerine bacterial classification of Leuconostoc mesenteroides dgnkzx002Leuconostoc Mesenteroidesdgnkzx002 is pressed 2% inoculum size in 20mLMRS liquid nutrient medium, and activation culture 20 hours are left standstill in 37 DEG C of incubators, obtain seed liquor;
S12. enlarged culturing: the seed liquor activated is inoculated in 1000mLMRS liquid nutrient medium by 5% inoculum size, and in 37 DEG C of incubators quiescent culture 20 hours;
S13. thalline suspension preparation: after having fermented, fermentation liquor 9500r/min, 4 DEG C of centrifugal 15min collect thalline, wash 2 times, the viable bacteria body needed for centrifugal acquisition by stroke-physiological saline solution.Finally with appropriate physiological saline thalline hanged and made cell suspending liquid.
MRS liquid culture based component: 1L distilled water, peptone 10g, extractum carnis 10g, yeast extract paste 5g, dipotassium hydrogen phosphate 2g, dibasic ammonium citrate 2g, sodium acetate 5g, glucose 20g, magnesium sulfate heptahydrate 0.58g, four water manganous sulfate 0.25g, tween 80 1mL, pH6.2 ~ 6.4,121 DEG C of sterilizing 15min.
S2. the preparation of preserving fungus agent: preserving fungus agent is containing, for example the component of lower mass percent: 1% chitosan, 2% citric acid, 0.85% sodium-chlor, 0.5% kojic acid and 2% Sodium.alpha.-hydroxypropionate, surplus is water, cell concentration 2.9 × 10
8cFU/mL.First take kojic acid, sodium-chlor and citric acid by formula to mix, add suitable quantity of water be heated to 60 ~ 70 DEG C and stir, it is made to dissolve completely, (Ke glycan takes off acetyl Du≤95% slowly to add the chitosan weighed up by mass percentage in the solution while stirring, viscosity 100 ~ 200mPa.s), treat that chitosan is fully swelling, solution adds Sodium.alpha.-hydroxypropionate after being cooled to room temperature, finally add thalline suspension and residue water, make cell concentration in preservation agent be 2.9 × 10
8cFU/mL.
S3. be that object is tested with Litchi chinensis cv. Nuomici.Pluck litchi fruits morning at fine day, choose healthy homogeneous without disease and pest, size, have no mechanical damage, the litchi fruits of about 8 maturations; To lichee precooling 2h under 5 DEG C of conditions, 20 DEG C of air-conditioned room cold wind dry up surface-moisture; Germicidal treatment is carried out with ultra violet lamp 30min; Soak litchi fruits 3min with above-mentioned preservation agent, cold wind dries up, with hamper splendid attire, and at the inner place mat wrapping paper of hamper in order to avoid Fructus Litchi is caused brown stain by globule dipping, 5 DEG C, preserve under relative humidity 80% ~ 90% condition.After the storage period of 25 days, experimental group healthy fruit can reach 86.7% and control group only has 20%.
Reference examples choose healthy homogeneous without disease and pest, size, have no mechanical damage, the litchi fruits of about 8 maturations.To lichee precooling 2h under 5 DEG C of conditions, cold wind dries up surface-moisture.Germicidal treatment is carried out with ultra violet lamp 30min.With hamper splendid attire, and at the inner place mat wrapping paper of hamper in order to avoid Fructus Litchi is caused brown stain by globule dipping, 5 DEG C, preserve under relative humidity 80% ~ 90% condition.Experimental result is shown in shown in accompanying drawing 10.
The application experiment of embodiment 4 bacterial strain
S1. the cultivation of fresh-keeping bacterium:
S11. the activation of bacterial strain: fresh-keeping bacterial strain is pressed 2% inoculum size in 20mLMRS liquid nutrient medium by glycerine pipe, and leave standstill activation culture 20 hours in 37 DEG C of incubators;
MRS liquid culture based component: 1L distilled water, peptone 10g, extractum carnis 10g, yeast extract paste 5g, dipotassium hydrogen phosphate 2g, dibasic ammonium citrate 2g, sodium acetate 5g, glucose 20g, magnesium sulfate heptahydrate 0.58g, four water manganous sulfate 0.25g, tween 80 1mL, pH6.2 ~ 6.4,121 DEG C of sterilizing 15min.
S12. enlarged culturing: by the seed liquor activated by 5% inoculum size be inoculated in 1000mLMRS liquid nutrient medium, and in 37 DEG C of incubators quiescent culture 20 hours;
S13. thalline suspension preparation: after having fermented, fermentation liquor 8500r/min, 4 DEG C of centrifugal 15min collect thalline, wash 2 times, the viable bacteria body needed for centrifugal acquisition by stroke-physiological saline solution.Finally with appropriate physiological saline thalline hanged and made cell suspending liquid, cell concentration 2.4 × 10
8cFU/mL.
S2. be that examination material is tested with longan.Pluck longan fruit morning at fine day, choose healthy homogeneous without disease and pest, size, have no mechanical damage, the longan fruit of about 8 maturations; Transport laboratory back rapidly, carry out germicidal treatment with ultra violet lamp 30min; With above-mentioned bacterium liquid even application at longan branch fruit surface, with bubble chamber splendid attire after normal temperature dries, and at the inner place mat newspaper of case, in order to avoid Fructus Litchi is caused brown stain by the globule dipping that respiration produces on tank wall, preserve under 29 ~ 32 DEG C of hot and humid conditions with relative humidity 97% ~ 99%, duration of storage can observe experimental group color significantly better than control group.
Reference examples:
Choose healthy homogeneous without disease and pest, size, have no mechanical damage, the longan fruit of about 8 maturations; Germicidal treatment is carried out with ultra violet lamp 30min; With clear water and P applied levels even application on litchi fruits surface, with the bubble chamber splendid attire of liner newspaper after normal temperature dries, preserve under the hot and humid conditions of 29 ~ 32 DEG C and relative humidity 97% ~ 99%.
Result is as shown in accompanying drawing 11 ~ 14.Fig. 11 illustrates each process healthy fruit, clear water process healthy fruit is 13.33%, and P applied levels process healthy fruit 40%, dgnkzx002 process healthy fruit is 43.33%.Fig. 12 illustrates PPO and suppress result, curve 1 is clear water process, and curve 2 is P applied levels process, and curve 3 is dgnkzx002 process.Fig. 13 shows POD and suppress result, curve 1 is clear water process, and curve 2 is P applied levels process, and curve 3 is dgnkzx002 process.Figure 14 shows SOD and keep result, curve 1 is clear water process, and curve 2 is P applied levels process, and curve 3 is dgnkzx002 process.
Embodiment 5 application experiment
Apply preserving fungus agent of the present invention and carry out fresh-keeping test to longan, the preparation of preserving fungus agent is with reference to embodiment 3.
Test materials: longan.
Test method: fine day plucks longan fruit morning, transports laboratory back for subsequent use.Preservation agent experimental group of the present invention, clear water treatment group and P applied levels process are set respectively, three groups are all selected that size is even, color and luster is consistent, anosis worm, undamaged healthy fruit test, three repetitions, each repetition 1 kilogram.Each group is soaked longan fruit 1 minute with preserving fungus agent of the present invention, clear water and P applied levels respectively, dries.Being dispensed in plastic basket, is 29 ~ 32 DEG C and relative humidity 97% ~ 99% time storage in room temperature, the investigation of 7 days laggard row healthy fruit, reducing sugar content and Vitamin C contents.Experimental result is asked for an interview shown in accompanying drawing 15 ~ 23.In accompanying drawing 17, curve 1 is clear water process, and curve 2 is P applied levels process, and curve 3 is preservation agent process of the present invention.In accompanying drawing 18, curve 1 is clear water process, and curve 2 is P applied levels process, and curve 3 is preservation agent process of the present invention.In accompanying drawing 19, curve 1 is clear water process, and curve 2 is P applied levels process, and curve 3 is preservation agent process of the present invention.In accompanying drawing 20, curve 1 is clear water process, and curve 2 is P applied levels process, and curve 3 is preservation agent process of the present invention.In accompanying drawing 21, curve 1 is clear water process, and curve 2 is P applied levels process, and curve 3 is preservation agent process of the present invention.In accompanying drawing 22, curve 1 is clear water process, and curve 2 is P applied levels process, and curve 3 is preservation agent process of the present invention.In accompanying drawing 23, curve 1 is clear water process, and curve 2 is P applied levels process, and curve 3 is preservation agent process of the present invention.
Experimental result is summed up, and longan peel polyphenoloxidase (PPO) and peroxidase (POD) activity of preservation agent process of the present invention are all significantly less than clear water, P applied levels process, and superoxide-dismutase (SOD) activity obtains good maintenance.And, preservation agent process of the present invention effectively can delay the reduction of pulp total reducing sugar, reducing sugar, vitamins C (Vc), soluble solid (TSS) and titratable acid (TA) content, keep taste of fruit, prevent saprophytic microorganism from infecting, improve the commodity rate (healthy fruit (%)) of longan fruit.
Claims (5)
1. a strain Leuconostoc mesenteroides (
leuconostoc Mesenteroides) bacterial strain, described bacterial strain is preserved in China typical culture collection center on November 26th, 2012, and preserving number is CCTCC M 2012479; Described Leuconostoc mesenteroides obviously can reduce polyphenoloxidase and the Peroxidase activity of lichee or longan, keeps lichee or longan peel superoxide dismutase activity.
2. bacterial strain according to claim 1, it is characterized in that, the colonial morphology of described bacterial strain is: circular, creamy-white, and convex growth is moistening.
3. a cultural method for bacterial strain described in claim 1 or 2, is characterized in that, be by the glycerine pipe freezing bacterial classification of described bacterial strain by 2% inoculum size in MRS liquid nutrient medium, activation culture obtains seed liquor; By seed liquor by 5% inoculum size receive in MRS liquid nutrient medium, cultivation and fermentation obtains fermented liquid, by fermentation liquor collected by centrifugation thalline, after stroke-physiological saline solution washing, centrifugal and get final product.
4. cultural method according to claim 3, is characterized in that, described MRS liquid culture based component: 1L distilled water, peptone 10g, extractum carnis 10g, yeast extract paste 5g, dipotassium hydrogen phosphate 2g, dibasic ammonium citrate 2g, sodium acetate 5g, glucose 20g, magnesium sulfate heptahydrate 0.58g, four water manganous sulfate 0.25g, tween-80 1mL, pH6.2 ~ 6.4,121 DEG C of sterilizing 15min.
5. cultural method according to claim 3, is characterized in that, described by fermentation liquor collected by centrifugation thalline be through 8000r/min, 4 DEG C centrifugal.
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